A physiologically active substance such as protein, peptide, hormone and a metabolite thereof is a substance carrying information transmission to adjust action of a specific organ in vivo, and thus fulfilling an important role in life activity. Therefore, it is important to inspect concentration of the physiologically active substance in a specific disease. For example, there has been reported that there is relation between increase and decrease in estrogen, which is a female hormone, and risk of breast cancer. The physiologically active substance exerts action thereof in quite a small amount, and concentration in a body is very low level of pg/mL to ng/mL. Accordingly, there has been required a method for quantifying the physiologically active substance in an organism sample, simply and accurately.
As one of the methods for quantifying the physiologically active substance, there is immunoassay. An automatic analysis device using the immunoassay enables to provide random access, and high throughput, therefore it has been used widely as a clinical laboratory test device (PATENT LITERATURE 2). For example, in the immunoassay, detection is performed by utilization of an antibody which specifically recognizes a subject component to be measured, for example, by capturing the subject component to be measured in a specimen using the antibody (a primary antibody), and then by utilizing a secondary antibody which further selectively captures the above primary antibody. On the other hand, it has been known generally that cross-reactivity is generated in the immunoassay. The cross-reactivity is a phenomenon where the primary antibody captures not only the subject component to be measured which the primary antibody should originally captures but also molecules having a similar structure, for example, a metabolite of the subject component to be measured or the like. In particular, the physiologically active substance is a low molecular weight substance, and has low specificity of antibody recognition, therefore the cross reaction appears.
In addition, application of mass spectrometry to clinical laboratory tests has also been prevailed, and it has been used as a method for quantifying the physiologically active substance. Because the mass spectrometry is measured based on the mass of a subject component, it is a measurement technology that identification is possible from, for example, a molecule having a similar structure of a metabolite or the like. An MRM (Multiple Reaction Monitoring) mode of a triple quadrupole mass spectrometer, having particularly high selectivity, is a method for passing through only a precursor ion at a quadrupole of the first stage, cleaving this ion at the next collision cell, and monitoring only a product ion specific to the compound at a quadrupole of the second stage. This method provides mass to charge information specific to a compound, and enables the relative quantification of the subject component to be measured, by correction with an internal standard substance which had been added in advance and has known concentration.
There has been disclosed PATENT LITERATURE 1 in which the antigen-antibody reaction and the mass spectrometry are applied, aiming at quantification of protein. In this method, firstly, by adding an enzyme to a specimen of serum or the like, protein is digested to peptide. Next, by adding and reacting antibody magnetic beads immobilized with an anti-peptide antibody and an internal standard substance labeled with a stable isotope to peptide which is a subject component to be measured, the peptide which is the subject component to be measured and the internal standard substance are bound to the antibody magnetic beads. After that, a treated specimen is introduced to a device which can concentrate the subject component to be measured and detect by mass spectrometry, and thus the quantification of peptide is performed. As for the composition of this device, tubing is connected via a plurality of valves at the former stage, and a magnet is arranged at the exterior side of a part of tubing. The concentrated peptide is ionized using an electrospray ionization method (ESI), to perform analysis using liquid chromatography/mass spectrometry (LC-ESI-MS). This device enables the concentration of the subject substance to be measured bound with the antibody magnetic beads, desorption from the antibody magnetic beads, and online detection using the mass spectrometer. In NON PATENT LITERATURE 1, analysis of peptide having physiologically active action is performed by applying the antigen-antibody reaction and the mass spectrometry. Protein, which is the subject substance to be measured, is made bound and concentrated by using polystyrene beads immobilized with the antibody. The analysis is performed using mass spectrometry (MS), by enzymatic digestion of the concentrated protein, cleaving it to peptide which is the subject substance to be measured, and ionizing using matrix-assisted laser desorption ionization (MALDI). In NON PATENT LITERATURE 2, although the steps of the antigen-antibody reaction and the enzymatic digestion are similar, analysis is performed by liquid chromatography/mass spectrometry (LC-ESI-MS) by ionization using the electro-spray ionization method (ESI). The steps up to the enzymatic digestion are processed, for example, in a 96-well plate, using an automatic pipette tool to enhance throughput.